1. Field of Invention
The present invention relates to improved coagulation assay reagents containing lanthanide compounds, and the assays performed therewith, particularly a protein C assay using a lanthanide containing reagent having surprising improvements in sensitivity of the assay.
2. Discussion of the Background
In this document, the term “coagulation assay” is used to denote a class of assays which includes (i) clotting or clot formation assays, (ii) clot lysis assays, and (iii) clotting parameter(s) assays.
Blood clotting reactions, in general, employed as clinical assays measure the time required for the formation of a fibrin clot. The most common of these reactions is the prothrombin time test.
Many approaches exist for measuring clot formation in prothrombin time assays. All of these clot formation based assays utilize thromboplastin to react with the patient's blood sample. Available approaches differ in the means used to detect when clot formation occurs. They may also differ in the types of apparatus used and in the constituents of and/or additives to the reagent.
Blood clotting assays are principally used for screening, diagnosis, and for monitoring patients receiving anticoagulant therapy. There are many types of coagulation assays. These include prothrombin time (PT), partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), fibrinogen assay, thrombin clotting time (TCT), activated clotting time (ACT), etc. The most frequently performed of these assays is prothrombin time.
A principal use of prothrombin time (PT) determinations is to monitor patients receiving oral anticoagulants such as warfarin. An accurate monitoring of coagulation in these patients is important to prevent recurrent clotting (thrombosis) and to keep the coagulation mechanism sufficiently active to prevent spontaneous bleeding. Prothrombin time testing provides information to permit better drug control to be achieved through the regulation of drug dosage.
In conventional practice, PT assays are performed by the addition of a liquid reagent to a plasma sample. The reagents are typically supplied in dried form and consist primarily of tissue thromboplastin and calcium chloride. The dried reagent is reconstituted before use by addition of a measured amount of distilled water.
These reagents are thermally sensitive, and refrigeration prior to use is required. The shelf life of the reagent in dried form is from one to two years. However, when it is reconstituted the reagent is considerably more labile and must be used within a few hours or discarded. In some cases reconstituted reagents can be kept for a few days under refrigeration.
Prothrombin time assays are performed by mixing sample and reagent at 37° C., and monitoring the progress of the reaction until a perceptible clot (or “gel clot”) is detected. The development of a gel clot is the end point of the reaction. This end point may be detected in various ways; by viscosity change, by electrode reaction, and, most commonly, by photometric means. The test result is generally compared to a result using a normal (control) plasma.
Before performing the test, a blood sample is collected in a tube or syringe containing anticoagulant (citrate). The blood sample is centrifuged, and the plasma separated (e.g., by decantation) from the red blood cells. A measured quantity (usually 0.1 ml) of plasma is pipetted into the reaction vessel or cuvette. A measured amount of reagent is then added manually via pipette or automatically by means of other volumetric delivery systems capable of metering a known, preset quantity of reagent. Alternatively, the sample can be added to the reagent directly.
Typically, 0.2 ml of reagent is employed. The addition of the reagent initiates the reaction. Many existing blood clotting assays, and in particular PT assays, all suffer from at least one of the following disadvantages: difficulty in performance, requirement of highly trained personnel, inaccuracy in measurement, reagent instability, large consumption of reagent, etc.
One solution to this problem was addressed in Oberhardt, U.S. Pat. No. 5,110,727, in which a dry reagent based reaction slide is provided for performing coagulation assays quickly, accurately and simply. Such tests are marketed by Pharmanetics, Inc.
The capacity of blood to clot, as well as to not clot, is dependent on a large number of factors and cofactors. The ability of central clinical laboratories to reliably and conveniently assay for Protein C in whole blood or plasma samples can be critical in monitoring individuals for whom an inappropriate coagulation episode is a life-threatening problem. The blood coagulation system is dominated by sequential proteolytic activation reactions of inactive precursors, called zymogens. Forward clotting reactions are controlled by simultaneous activation of anticoagulant zymogens that serve to limit the extent of clot formation and initiate the fibrinolytic system to resolve the clot. A crucial enzyme in the cascade is thrombin. Thrombin proteolyzes fibrinogen to form a clot from insoluble fibrin molecules. Thrombin is a poor activator of Protein C in solution. At the same point in the coagulation cascade, thrombin may bind to thrombomodulin on the endothelial cell surface and become a potent activator of Protein C but no longer have the ability to cleave fibrinogen to fibrin. Generation of low levels of thrombin favors binding to thrombomodulin and activation of Protein C. Activated Protein C proteolytically inactivates Factors Va and VIIIa to reduce the rate of thrombin formation.
Decreased levels and/or activity of Protein C is associated with an increased thrombotic risk. The thrombotic risk may present itself as a deep venous thrombosis or as disseminated intravascular coagulation (DIC). Broekmans, A. W. et al, N. Engl. J. Med., 309, 340-344 (1983). The concentration of Protein C has been correlated to these thrombotic events and to the severity of the event. Griffin J. H. et al, Blood, 60(1), 261-264 (1982); Marlar R. A. et al, Blood, 66(1), 59-63, (1985). There have been several reported uses of Protein C concentrates, purified Protein C and activated Protein C to treat and prevent DIC. Rintala E. et al, Lancet, 347, 1767 (1996); Smith, O. P. et al, Lancet, 350, 1590-1591 (1997); Favier R. et al, Hemat. Cell Ther., 40, 67-70 (1998).
FDA 510(k) approved Protein C assays can be broadly grouped into three categories: antigenic, chromogenic/amidolytic, and coagulometric. The antigenic assays include ELISA, EIA and RIA type tests. These assays do not determine if the protein is functional because the antibodies are not directed to epitopes associated with functional activity.
The chromogenic/amidolytic assays rely on the ability of the active site of the enzyme to cleave a small synthetic substrate to release an intensely colored product. There may exist a discrepancy between the activity of activated Protein C towards a synthetic substrate and towards a natural biological substrate. In addition, other functional characteristics of the enzyme (i.e. cofactor interaction) are not tested by the synthetic substrate.
Coagulometric assays performed in the central laboratory are cumbersome and are best suited to batch analysis. These assays require generation of a standard curve prior to sample analysis. The samples require an initial dilution, an incubation for activation of the Protein C, then initiation of the clotting cascade. The relationship between clot time and Protein C activity (expressed as percent of normal) established by the standard curve is used to interpolate the Protein C activity of the unknown sample. Although differences in laboratory procedure exist, a standard curve is routinely repeated with each group of patient samples tested, when new lots of reagent are opened or when the control does not fall within its prescribed range. The multiple manipulations required for determination of one sample provides an assay that is cumbersome and batch analysis is more economical in terms of technologist time and reagent costs.
There is thus a strongly felt need for a simple, facile and accurate method for the performance of blood clotting assays, e.g., in medical applications. Such a method should be based on a minimum number of manipulations of either a sample or reagent. Ideally such a method should be easily utilized by persons without extensive clinical laboratory training and should require no sample or reagent-containing solution preparation. It should not suffer the problems associated with reagent instability and be very accurate. It should permit effective mixing of sample and reagent. It should require only a very small amount of sample. And it should be able to perform automatic treatments of the sample, e.g., it should not require centrifugation of the blood sample or any other off line cell separation process. Available clotting parameter assays likewise suffer salient disadvantages.
Clotting parameter assays are referred to herein as function and structure-based assays in the broad realm of coagulation diagnostics which do not utilize clot formation or clot lysis processes to generate end points. Most of these assays utilize chromogenic synthetic substrates to quantify molecular markers or specific factors or components associated with coagulation. These are typically functional reaction based assays as opposed to most immunoassays which could detect the same molecules but utilize structure recognition and may therefore still identify inhibited components or defective components, neither of which may be functional. The present invention does not deal specifically with immunoassays but may be generally applicable to homogeneous chromogenic and fluorogenic immunoassays.